Protein Precipitation by Salts

 

 

PROTEIN PRECIPITATION BY SALTS Rüveyda AKÇİN, Gebze Technical University, Turkey  

Experiment 5 AIM The solubility of proteins in pure water is low. The neutral salt is added for more protein dissolution, and this is done protein precipitation. Virtually all proteins can be precipitated at high concentrations of certain salts. However, the salts that that are used in protein precipitations are not equally effective as protein precipitants and the concentrations required for precipitation will generally be different for different proteins. Accordingly, by judicious choice of experimental conditions, one can often o ften achieve purification of a protein by means of selective precipitation by salts. The purpose of this experiment is to acquaint you with this technique.

INTRODUCTION The solubility of protein depends on, among other things, the salt concentration in the solution. At low concentrations, the presence of salt stabilizes the various charged groups on

purification and concentration of large quantities of protein. Ammonium sulfate, (NH4)2SO4, is the most commonly used salt for salting out proteins because its large solubility

a protein molecule, thus attracting protein into the solution and enhancing the solubility of protein. This is commonly known as salting-in. However, as the salt concentration is increased, a point of maximum protein solubility is usually reached. Further increase in the salt concentration implies that there is less and less water available to solubilize protein. Finally, protein starts to precipitate when there are not sufficient water molecules to interact with protein molecules. This phenomenon of protein precipitation in the presence of excess salt is known as salting-out. Since proteins

in water, its relative freedom from temperature effects, and it has no harmful effects on most of the proteins. The most effective pH region for salting out of the desired protein is at its isoelectric point because the protein is least soluble when its net charge is zero.

precipitate at different salt concentrations, salting out is the basis of one of most commonly used protein purification procedures. Adjusting the salt concentration in a solution containing a mixture of proteins to  just below the precipitation point of the protein to be purified eliminates many unwanted proteins from the solution. Then, after removing the precipitated proteins by filtration or centrifugation, the salt concentration of the remaining solution is increased to precipitate the desired protein. The precipitation of desired protein is then dissolved in water to make a solution of this protein. This procedure results in a significant

(Effect of salt concentration on protein solubility) Intermolecular interactions between protein molecules can have different origins, such as electrostatic, hydrophobic, van der waals, and hydrogen bonding. It is difficult to pinpoint the exact relative contributions from each type of interaction to the (overall) protein-protein interactions. In this review, I focus on

 

explaining the modulations of electrostatic protein-protein interactions by the simple salt ions through their specific interactions (or binding) from both cation and anion with protein surface at salt concentrations below 0.5- 1 M. In addition, the complete picture of interactions may be better understood by considering the following biophysical properties of proteins and salt ions: (i) the net charge, surface charge density and hydrophobicity of a protein; (ii) hydration, size, polarizability and valency of salt ions. The discussion is based on the t he recent experimental results reported in literature and findings from Amgen using the following experimental techniques, such as protein solubility measurement, phase transition temperature of Tcritical  (critical temperature) or T cloud  (cloud temperature) for liquid-liquid phase separation

subsequent purification steps are possible. Dialysis is one of the common operations in biochemistry to separate dissolved molecules by passing through a semi-permeable membrane according to their molecular dimensions. Semi-permeable membrane is containing pores of less than macromolecular dimensions. These pores allow small molecules, such as those of solvents, salts, and small metabolites, to diffuse across the membrane but block the passage of larger molecules. Cellophane (cellulose acetate) is the most commonly used dialysis material although many other substances such as nitrocellulose and collodion are similarity employed. So, dialysis is a method in which an aqueous solution containing both macromolecules and very small molecules which are placed in a dialysis bag which is in tern placed in a large container of a given

and small angle X-ray scattering (SAXS). It has been demonstrated that there is a strong correlation between protein solubility and protein-protein interactions: protein solubility decreases when the protein-protein interactions become less repulsive or more attractive (for a protein for which its solubility increases with temperature). Also it is generally accepted that for a protein solution with an upper consolute point, an increase in phase transition temperature, as a result of change in the solution condition, indicates that proteinprotein interactions become less repulsive or

buffer or distilled water. Thus small solute molecules freely pass through the membrane, and after several hours of stirring the equilibrium will reach (the concentration inside and outside the bag are the same). Thus, at equilibrium the concentration of small molecules outside and inside the bag is the same while the macromolecules remain inside the bag. During dialysis the external fluid should be changed in order to reach the required composition inside the dialysis bag. There are three factors that affecting the rate of dialysis: the first is the concentration

more attractive.

differences of that molecules between the internal and external solution (which is the driving force for the movement of the molecules). The second is mixing on both sides si des of dialysis membrane will increase the rate of movement prevent the small particles on the side of low concentration. The third is dialyzable particles size versus pore size of the membrane, substances that are very much smaller than the pore size will reach equilibrium faster than substances that are only slightly smaller than the pores. The main point to be noted is that there is a rapid initial

salt

ion’s

effects

on

the

intermolecular

(Hofmeister Series) A solution containing the protein of interest often must be further altered before

drop in dialysis process followed by a slow approach to equilibrium. 

 

same general procedure used for the addition of ammonium sulfate. When the added salt fails to dissolve, allow the solution to stand for about a bout 10 minutes and then fitler. Save the filtrate.

RESULT Two replica are used to get an exact result.

  (Use of dialysis to separate small and large molecules.)

MATERİALS AND METHODS 

  25 ml egg white, 4 grams ammonium

Standart Conc.(mg/ml) Abs Rep 1 Abs Rep 2 Abs Average Abs Avr -Blank Avr 1.5 1.633 1.568 1.6005 1.57845 1 1.202 1.199 11..2005 1.17845

A B C

0.75

0.854

0.841

0.8475

0.82545

D

0.5

0.69

0.674

0.682

0.65995

E F Bl ank White Egg

0.25 0.125 0

0.401 0.222 0.0225 1.235

0.403 0.238 0.0216 1.24

0.402 0.23 0.02205 1.2375

0.37995 0.20795 0 1.21545



sulfate, 2 g solid sodium sulfate, 2 g solid sodium chloride vortex, pipet, 3 falcons, filter page, dialysis page.

The concentrations vs. absorbans graphic according to above datas;

1.  Obtain a 5 ml sample of 1/10 dilute egg white. To a 5 ml sample of the filtered dilute egg white, and sufficient solid ammonium sulfate to bring the solution to saturation with respect to this salt. To do this, slowly, with stirring, add the solid ammonium sulfate until it no longer goes into solution. Be sure to allow adequate time after each increment of ammonium sulfate is addd to determine whether it will dissolve. When the added salt fails to dissolve, allow the solution to stand for about 10 minutes and then fitler. Save the filtrate. 2.  To another 3 ml sample of the filtered dilute egg white, add sufficient solid sodium sulfate to bring the solution to saturation with respect to this salt. Use the same general procedure used for the addition of ammonium sulfate. When the added salt fails to dissolve, allow the solution to stand for about 10 minutes and then fitler. Save the filtrate. 3.  Finally, using a third 3 ml sample of the filtered dilute egg white, add suficien solid sodium chloride to bring the solution to saturation with respect to this salt. Use the

The following table contains solutions of different salts. Sa Salts lts

Vbe Vbefo fore re (m (ml) l) Va Vafte fterr (m (ml) l) Abs RRep ep 1 Abs Rep Rep 2 Abs Aver Averag agee Abs Avr Avr-B -Bla lank nk Av Avrr

NaCI

3

8

0.564

0.564

0.564

0.54195

Na2SO4

3

8

0.357

0.364

0.3605

0.33845

(NH4)2SO4

3

8.1

0.287

0.284

0.2855

0.26345

Calculation:  Knowing the initial and final volumes of the ammonium sulfate ( as well as sodium sulfate and sodium chloride filtrates) correct the

 

concentration of this following equation :

filtrate

using

the

Ccorrect= Ccorrect ×(Vbefore/Vafter) 

=100x [(1.1050-0.05407)/1.1050] =%95.10 According to information and calculations:

Where the volumes (V) are before and after dialysis. Finally , calculate the effectiveness of each salt as a protein precipitating agent using the following equation: %protein precipitated = 100×(Coriginal-Ccorrect)/Coriginal

where Coriginal is the concentration of protein in the original, filtreted egg white.

  For original diluted white egg;



DISCUSSION

y=1.21545 1.21545=0.991x+0.1205 Coriginal=1.1050 mg/ml

  For NaCI;



y=0.54195 0.54195=0.991x+0.1205 Cobserved=0.4253 mg/ml Ccorrect=0.4253x(3/8)=0.15949 mg/ml =100x [(1.1050-0.15949)/1.1050] =%85.57

  For Na2SO4;



y=0.33845 0.33845=0.991x+0.1205 Cobserved =0.2199 mg/ml Ccorrect= 0.2199x(3/8)=0.0824 mg/ml =100x[(1.1050-0.0824)/1.1050] =%92.54

  For (NH4)2SO4;

The distribution of hydrophilic and hydrophobic groups in the protein molecule affects the solubility of the proteins in various solvents. Using these properties of the proteins, precipitation processes are carried out in different forms according to the purpose. The most preferred of these precipitation processes is precipitation with high salt concentrations. According to this information, we performed sedimentation with three different salts in the experiment and measured the absorbance values. Each of the falcons of we tagged have 5 ml white egg and we added salts to these falcons until protein collapses we do not know how much protein the proteins will collapse, so we added salt to the protein precipitate. The solubility of proteins in water is low. Therefore we added high concentration salt. In addition proteins have an amino and an carboxyl, so it could be solved and then it have to ionized to precipitate. On the other hand, the reason reason for dialysis is separate to other ions from proteins. Thus, we get the proteins more pure. The most important point in dialysis, we have to change water every two days for better results.

REFERENCES



y=0.26345 0.26345=0.991x+0.1205

Jakoby, W.B., Crystallization as a purification technique, Enzyme Purification and Related Techniques, in Methods in Enzymology, Vol. 22, Jakoby, W.B., Ed., Academic Press, 1971.  

Cobserved =0.1442 mg/ml Ccorrect=0.1442x(3/8)=0.05407 mg/ml

Burgess, R. R. (1991). The use of polyethyleneimine in the purification of DNA

 

binding proteins. Meth. Enzymol. 208, 3 –10. Burgess, R. R., and Jendrisak, J. J. (1975). A procedure for the rapid, large-scale purification of E. coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14, 46344638.